Crystallized oxalate decarboxylase and methods of use

ABSTRACT

Pharmaceutical compositions comprising spray-dried oxalate decarboxylase crystals are disclosed. Methods to treat a disorder associated with elevated oxalate concentration using compositions comprising spray-dried oxalate decarboxylase crystals are also disclosed.

Oxalic acid is a dicarboxylic acid of the formula HO₂C—CO₂H. Oxalic acidexists primarily as oxalate in biological organisms, which is the saltform of oxalic acid. Oxalate is found in foods, such as, e.g., spinach,rhubarb, strawberries, cranberries, nuts, cocoa, chocolate, peanutbutter, sorghum, and tea. Oxalate is also a metabolic end product inhumans and other mammals. It is excreted by the kidneys into the urine.When combined with calcium, oxalic acid produces an insoluble product,calcium oxalate, which is the most prevalent chemical compound found inkidney stones.

Because mammals do not synthesize enzymes that degrade oxalate, oxalatelevels in an individual are normally held in check by excretion and lowabsorption of dietary oxalate. Elevated concentrations of oxalate areassociated with a variety of pathologies, such as primary hyperoxaluria,enteric hyperoxaluria, and idiopathic hyperoxaluria. Leumann et al.,Nephrol. Dial. Transplant. 11:2556-2558 (1999) and Earnest, Adv.Internal Medicine 24:407-427 (1979). Increased oxalate can be caused byconsuming too much oxalate from foods, by hyperabsorption of oxalatefrom the intestinal tract, and by abnormalities of endogenous oxalateproduction. Hyperabsorption of oxalate in the colon and small intestinecan be associated with intestinal diseases, including hyperabsorptioncaused by diseases of bile acid and fat malabsorption; Heal resection;and, for example, by steatorrhea due to celiac disease, exocrinepancreatic insufficiency, intestinal disease, and liver disease.

Hyperoxaluria, or increased urinary oxalate levels, is associated with anumber of health problems related to the deposit of calcium oxalate inthe kidney tissue (nephrocalcinosis) or urinary tract (e.g., kidneystones, urolithiasis, and nephrolithiasis), Calcium oxalate may also bedeposited in, e.g., the eyes, blood vessels, joints, bones, muscles,heart and other major organs, causing damage to the same, See, e.g.,Leumann et al., J. Am. Soc. Nephrol. 12:1986 1993 (2001) and Monica etal., Kidney International 62:392 400 (2002). The effects of increasedoxalate levels can appear hi a variety of tissues. For example, depositsin small blood vessels cause painful skin ulcers that do not heal,deposits in bone marrow cause anemia, deposits in bone tissue causefractures or affect growth in children, and calcium oxalate deposits inthe heart cause abnormalities of heart rhythm or poor heart function.

Existing methods to treat elevated oxalate levels are not alwayseffective and intensive dialysis and organ transplantation may berequired in many patients with primary hyperoxaluria. Existing therapiesfor various hyperoxalurias include high-dose pyridoxine, orthophosphate,magnesium, iron, aluminum, potassium citrate, cholestyramine, andglycosaminoglycan treatment, as well as regimes for adjusting diet andfluid intake, for dialysis, and for surgical intervention, such as renaland liver transplantation. These therapies (e,g., low-oxalate or low-fatdiet, pyridoxine, adequate calcium, and increased fluids), are onlypartially effective and they may have undesirable adverse side effects,such as the gastrointestinal effects of orthophosphate, magnesium, orcholestyramine supplementation d the risks of dialysis and surgery.Accordingly, methods that safely remove oxalate from the body areneeded. Moreover, methods that degrade oxalate to reduce oxalate levelsin a biological sample are advantageous over a therapy, for example,that solely blocks absorption or increases clearance of oxalate.

The present disclosure relates to pharmaceutical compositions ofspray-dried oxalate decarboxylase (“OXDC”) crystals and their uses totreat oxalate-associated disorders, e.g., hyperoxaluria. In someembodiments, pharmaceutical compositions comprising spray-driedcrystalline oxalate decarboxylase can be administered to a mammal, e.g.,orally or directly to the stomach, to reduce oxalate levels and/or toreduce damage caused by calcium oxalate deposits in the mammal.

The invention is based in part on the discovery that a crystalline formof oxalate decarboxylase can be spray-dried without destroying theprotein crystals or their beneficial properties. It has previously beenunderstood by those of skill in the art that protein crystals could notbe spray-dried because the crystals would clog the spray-dryer duringthe manufacturing process. It was also believed that even if thespray-dryer could be adapted to process protein crystals, the crystalswould be fractured and broken down to a dust during processing,essentially eliminating any advantages associated with the crystallineform. Thus, it is surprising and unexpected that pharmaceuticalcompositions of the invention comprise spray-dried oxalate decarboxylasecrystals that retain their crystalline integrity, stability, andenzymatic activity, as well as providing the traditional advantagesassociated with spray-dried formulations.

The spray-dried oxalate decarboxylase crystal compositions of thepresent disclosure allow for increased activity per unit of weight orvolume. For example, the compositions allow for increased density (i.e.,increased weight per unit of volume), which enables one to fill moreunits of oxalate decarboxylase per unit of volume. In particularembodiments, spray-dried oxalate decarboxylase crystal compositions ofthe invention allow more units to be filled into a capsule of a givensize. Accordingly, spray-dried oxalate decarboxylase crystalcompositions of the present disclosure allow a therapeutically effectivedose to be delivered orally, rather than by infusion or injection, andallow for a reduced pill burden. The spray-dried oxalate decarboxylasecrystal compositions of the invention also improve stability, Withoutwishing to be bound by theory, it is believed that increased densitycontributes to improved stability. The spray-dried oxalate decarboxylasecrystal compositions of the invention allow for manufacturingimprovements, including improved stability and reduced waste fromfilling the spray-dried crystals directly into capsules as opposed toformulating with excipients, potentially reducing cost and decreasingproduction time.

The present disclosure further provides pharmaceutical compositionscomprising spray-dried oxalate decarboxylase crystals that can deliver ahigher level of activity into an oral dosage form of a given size. Forexample, in some embodiments, a size 000 capsule comprises at least11,000 units (U), at least 22,000 U, at least 33,000 U, at least 44,000U, at least 55,000 U, at least 66,000 U, or at least 77,000 U ofspray-dried oxalate decarboxylase crystals (OXDC crystals). In someembodiments, a size 000 capsule comprises between 11,000 and 77,000 U ofspray-dried OXDC crystals. In some embodiments, a size 000 capsulecomprises between 22,000 and 77,000 U of OXDC crystals. In someembodiments, a size 000 capsule comprises between 33,000 and 77,000 U ofspray-dried OXDC crystals. In some embodiments, a size 000 capsulecomprises between 44,000 and 77,000 U of spray-dried OXDC crystals. Insome embodiments, a size 000 capsule comprises between 55,000 and 77,000U of spray-dried OXDC crystals. In some embodiments, a size 000 capsulecomprises between 66,000 and 77,000 U of spray-dried OXDC crystals.

In some embodiments, a size 00 capsule comprises at least 8,000 U, atleast 15,000 U, at least 23,000 U, at least 30,000 U, at least 38,000 U,at least 46,000 U, or at least 53,000 U of spray-dried OXDC crystals. Insome embodiments, a size 00 capsule comprises between 8,000 and 53,000 Uof spray-dried OXDC crystals. In some embodiments, a size 00 capsulecomprises between 15,000 and 53,000 U of spray-dried OXDC crystals. Insome embodiments, a size 00 capsule comprises between 23,000 and 53,000U of spray-dried OXDC crystals. In some embodiments, a size 00 capsulecomprises between 30,000 and 53,000 U of OXDC crystals. In someembodiments, a size 00 capsule comprises between 38,000 and 53,000 U ofspray-dried OXDC crystals, In some embodiments, a size 00 capsulecomprises between 46,000 and 53,000 U of spray-dried OXDC crystals.

In some embodiments, a size 0 capsule comprises at least 5,000 U, atleast 11,000 U, at least 16,000 U, at least 22,000 U, at least 27,000 U,at least 33,000 U, or at least 38,000 U of OXDC crystals. In someembodiments, a size 0 capsule comprises between 5,000 and 38,000 U ofspray-dried OXDC crystals. In some embodiments, a size 0 capsulecomprises between 11,000 and 38,000 U of spray-dried OXDC crystals. Insome embodiments, a size 0 capsule comprises between 16,000 and 38,000 Uof OXDC crystals. In some embodiments, a size 0 capsule comprisesbetween 22,000 and 38,000 U of spray-dried OXDC crystals. In someembodiments, a size 0 capsule comprises between 27,000 and 38,000 U ofspray-dried OXDC crystals. In sonic embodiments, a size 0 capsulecomprises between 33,000 and 38,000 U of spray-dried OXDC crystals,

In some embodiments, a size 1 capsule comprises at least 3,000 U, atleast 6,000 U, at least 9,000 U, at least 12,000 U, at least 15,000 U,at least 18,000 U, or at least 21,000 U of spray-dried OXDC crystals. Insome embodiments, a size 1 capsule comprises between 3,000 and 21,000 Uof spray-dried OXDC crystals. In some embodiments, a size 1 capsulecomprises between 6,000 and 21,000 U of spray-dried OXDC crystals. Insome embodiments, a size 1 capsule comprises between 9,000 and 21,000 Uof spray-dried OXDC crystals, In some embodiments, a size 1 capsulecomprises between 12,000 and 21,000 U of spray-dried OXDC crystals. Insome embodiments, a size 1 capsule comprises between 15,000 and 21,000 Uof spray-dried OXDC crystals. In some embodiments, a size 1 capsulecomprises between 18,000 and 21,000 U of spray-dried OXDC crystals.

In all cases a unit is defined as the amount of spray-dried oxalatedecarboxylase crystals that will degrade one micromole of oxalate perminute at 37° C.

In a related aspect, the present disclosure features pharmaceuticalcompositions comprising spray-dried oxalate decarboxylase crystals whichare substantially active and stable in variable pH conditions (e.g.,about pH 2-9, about pH 2-7, or about pH 4-7), and/or in the presence ofa protease, e.g., one or more of, e.g., pepsin, chymotrypsin, orpancreatin. In some embodiments, the pharmaceutical composition retainsan activity at least 2-, 3-fold higher than the activity retained by anon-crystalline oxalate decarboxylase in acidic conditions (e.g., anacidic pH of about 2 to 3) and in the presence of a protease, asdescribed herein. In other embodiments, the pharmaceutical compositionis at least 200%, 300%, 400% more stable than a non-crystalline oxalatedecarboxylase in acidic conditions (eg., an acidic pH of about 2 to 3)and in the presence of a protease, as described herein. In someembodiments, the oxalate decarboxylase crystals are spray-driedcross-linked crystals. In some embodiments, the spray-dried oxalatedecarboxylase crystals are not cross-linked.

In some embodiments, the spray-dried crystals include oxalatedecarboxylase having a sequence identical or substantially identical toan oxalate decarboxylase sequence found in a natural source, such as aplant, bacterium and fungus, in particular from Bacillus subtilis,Collybia velutipes or Flammulina velutipes, Aspergillus niger,Pseudomonas sp. Synechocystis sp. Streptococcus mutans, Trameteshirsute, Sclerotinia sclerotiorum, T. versicolor, Postia placenta,Myrothecium verrucaria, Agaricus bisporus, Methylobacteriurraextorquens, Pseudomonas oxalaticus, Ralstonia eutrapha, Cupriavidusoxalaticus, Wautersia sp., Oxalicibacterium flavum, Ammoniiphilusoxalaticus, Vibrio oxalaticus, A. oxalativorans, Variovorax paradoxus,Xanthobacter autotrophicus, Aspergillus sp., Penicillium sp., and Mucorspecies. In other embodiments, the oxalate decarboxylase isrecombinantly produced.

In some embodiments, the cross-linked or uncross-linked crystals used inthe compositions of the invention include oxalate decarboxylase having asequence identical or substantially identical to the oxalatedecarboxylase sequence from Bacillus subtilis (SEQ ID NO:1). In someembodiments, the oxalate decarboxylase comprises a modification of thecysteine residue at position 383 (“C383”) of SEQ ID NO:1. In someembodiments, the modification comprises reaction of C383 with a thiolprotecting group. In some embodiments, the oxalate decarboxylasecomprises a sequence identical to SEQ ID NO:1, except that the cysteineresidue at position 383 has been substituted with a different aminoacid. In some embodiments, the oxalate decarboxylase comprises asequence identical to SEQ ID NO:1, except that C383 has been deleted. Inother embodiments, the oxalate decarboxylase comprises a sequenceidentical to SEQ ID NO:1, except that a C-terminal cysteine has beenadded to allow formation of an intramolecular disulfide ring. Becausenative or wild type oxalate decarboxylase may form a complex mixture ofmultimers, modification of C383 to reduce or eliminate the reactivethiol group can render the modified oxalate decarboxylase better suitedfor commercial scale of production. In one embodiment, protection of theC383 thiol group is carried out by cysteinylation.

In one aspect, the invention provides a method of reducing oxalateconcentration in a subject by administering a pharmaceutical compositionthat comprises spray-dried cross-linked or uncross-linked oxalatedecarboxylase crystals that are spray-dried as disclosed herein. Inanother aspect, the invention provides for use of any of the spray-driedcross-linked or uncross-linked oxalate decarboxylase crystalpharmaceutical compositions described herein in a method of reducingoxalate concentration in a subject. Administration of the pharmaceuticalcomposition can cause a reduction of oxalate concentration by at least10%, at least 20%, at least 30%, or at least 40% or more.

In some embodiments, the composition is administered orally or via anextracorporeal device. In one embodiment, the extracorporeal device is acatheter, e.g., a catheter coated with spray-dried oxalate decarboxylasecrystals. In other embodiments, the composition is administered as asuspension, dry powder, capsule, or tablet. In one embodiment, themethod of reducing oxalate concentration in a mammal includes a step ofassaying the oxalate concentration in a biological sample of the mammal,such as a urine, blood, plasma, or serum sample.

In another aspect, the present disclosure provides a method of treating,preventing, and/or slowing the progression of a disorder associated withelevated oxalate concentrations in a mammal by administeringpharmaceutical compositions comprising spray-dried cross-linked oruncross-linked oxalate decarboxylase crystals to the mammal. In oneembodiment, the disorder associated with elevated oxalate concentrationis a kidney disorder, joint disorder, eye disorder, liver disorder,gastrointestinal disorder, or pancreatic disorder. In certainembodiments, the disorder is primary hyperoxaluria, enterichyperoxaluria, idiopathic hyperoxaluria, ethylene glycol poisoning,cystic fibrosis, inflammatory bowel disease, urolithiasis,nephrolithiasis, chronic kidney disease, hemodialysis, gastrointestinalbypass, and kidney stones.

The details of one or more embodiments of the present disclosure are setforth in the accompanying drawings and the description below.

DESCRIPTION OF DRAWINGS

FIG. 1 is the pH activity profile of non cross-linked vs. cross-linkedOXDC crystals (CLEC).

FIG. 2 is the pH stability profile of oxalate decarboxylase crystals.

FIG. 3 is the pH stability profile of non cross-linked OXDC crystals vs.cross-linked (CLEC) OXDC in simulated gastric fluid with pepsin at pH3.7.

FIG. 4 is the accelerated stability profile for lyophilized vs.spray-dried oxalate decarboxylase crystals.

FIG. 5 is the stability data showing activity of OXDC crystalsspray-dried with various excipients.

DETAILED DESCRIPTION OF EMBODIMENTS

The present disclosure is based, in part, on the discovery that crystalsof oxalate decarboxylase (OXDC) can be spray-dried for use inpharmaceutical compositions. These pharmaceutical compositionscomprising spray-dried cross-linked or uncross-linked OXDC crystals andmethods of administering them to treat the symptoms of hyperoxaluria andother oxalate-related disorders in a mammal are described herein.

Definitions

As used herein, a “biological sample” is biological material collectedfrom cells, tissues, organs, or organisms, for example, to detect ananalyte. Exemplary biological samples include a fluid, cell, or tissuesample. Biological fluids include, for example, serum, blood, plasma,saliva, urine, or sweat. Cell or tissue samples include biopsy, tissue,cell suspension, or other specimens and samples, such as clinicalsamples.

A “crystal” is one form of the solid state of matter, comprising atomsarranged in a pattern that repeats periodically in three dimensions(see, e.g., Barret, Structure of 25 Metals, 2nd ed., McGraw-Hill, NewYork (1952)). A crystal form of a polypeptide, for example, is distinctfrom a second form—the amorphous solid state. Crystals displaycharacteristic features including shape, lattice structure, percentsolvent, and optical properties, such as, ag., refractive index. An OXDCcrystal may be cross-linked as described in U.S. Pat. No. 8,142,775, oruncrosslinked.

An “extracorporeal device”is a structure that is not within the body forbringing a body fluid in contactwithspray-dried OXDC crystals in thetreatment of an individual. Preferably, an extracorporeal device is adevice used for dialysis, including kidney dialysis, a device forcontinuous arteriovenous hemofiltration, an extracorporeal membraneoxygenator, or other device used to filter waste products from thebloodstream. Similarly, components of devices to filter waste productsare encompassed by the term, including a tube, a porous material, or amembrane, for example. In particular, an extracorporeal device may be adialysis device. It may also be a membrane of a dialysis device.

A “functional fragment” of OXDC is a portion of an OXDC polypeptide thatretains one or more biological activities of OXDC, such as the abilityto catalyze the decarboxylation of oxalate. As used herein, a functionalfragment may comprise terminal truncations from one or both termini,unless otherwise specified. For example, a functional fragment may have1, 2, 4, 5, 6, 8, 10, 12, 15, or 20 or more residues omitted from theamino and/or carboxyl terminus of an OXDC polypeptide. Preferably, thetruncations are not more than 20 amino acids from one or both termini, Afunctional fragment may optionally be linked to one or more heterologoussequences.

The term “individual” or “subject” refers to any mammal, including butnot limited to, any animal classified as such, including humans, nonhuman primates, primates, baboons, chimpanzees, monkeys, rodents (e.g.,mice, rats), rabbits, cats, dogs, horses, cows, sheep, goats, pigs, etc.

The term “isolated” refers to a molecule that is substantially free ofits natural environment. For instance, an isolated protein issubstantially free of cellular material or other proteins from the cellor tissue source from which it is derived. The term refers topreparations where the isolated protein is sufficiently pure to beadministered as a therapeutic composition, or at least 70% to 80% (w/w)pure, more preferably, at least 80% 90% (w/w) pure, even morepreferably, 90 to 95% pure; and, most preferably, at least 95%, 96%,97%, 98%, 99%, 99.5%, 99.8% or 100% (w/w) pure.

As used herein, the term “about” refers to up to ±10% of the valuequalified by this term. For example, about 50 mM refers to 50 mM ±5 mM;about 4% refers to 4%±0.4%.

As used herein, “oxalate-associated disorder” refers to a disease ordisorder associated with pathologic levels of oxalic acid or oxalate,including, but not limited to hyperoxaluria, primarily hyperoxaluria,enteric hyperoxaluria, idiopathic hyperoxaluria, ethylene glycol(oxalate) poisoning, idiopathic urinary stone disease, renal failure 5(including progressive, chronic, or end-stage renal failure),steatorrhoea, malabsorption, ileal disease, vulvodynia, cardiacconductance disorders, inflammatory bowel disease, cystic fibrosis,exocrine pancreatic insufficiency, Crohn's disease, ulcerative colitis,nephrocalcinosis, urolithiasis, and nephrolithiasis, Such conditions anddisorders may optionally be acute or chronic. Oxalate-associateddisorders associated with kidneys, bone, liver, gastrointestinal tract,and pancreas are known in the art. Further, it is well known thatcalcium oxalate can deposit in a wide variety of tissues including, butnot limited to, the eyes, blood vessels, joints, bones, muscles, heart,and other major organs leading to a number of oxalate-associateddisorders.

“Oxalic acid” exists predominantly in its salt form, oxalate (as saltsof the corresponding conjugate base), at the pH of urine and intestinalfluid (pK_(a1)=1.23, pK_(a2)=4.19). Earnest, Adv. Internal Medicine24:407 427 (1979). The terms “oxalic acid” and “oxalate” are usedinterchangeably throughout this disclosure. Oxalate salts comprisinglithium, sodium, potassium, and iron (II) are soluble, but calciumoxalate is typically very poorly soluble in water (for example,dissolving only to about 0.58 mg/100 ml at 18° C. Earnest, Adv. InternalMedicine 24:407 427 (1979)). Oxalic acid from food is also referred toas dietary oxalate. Oxalate that is produced by metabolic processes isreferred to as endogenous oxalate. Circulating oxalate is the oxalatepresent in a circulating body fluid, such as blood.

The terms “therapeutically effective dose,” or “therapeuticallyeffective amount,” refer to that amount of a compound that results inprevention, delay of onset of symptoms, or amelioration of symptoms ofan oxalate-related condition, including hyperoxaluria, such as primaryhyperoxaluria or enteric hyperoxaluria. A therapeutically effectiveamount will, for example, be sufficient to treat, prevent, reduce theseverity, delay the onset, and/or reduce the risk of occurrence of oneor more symptoms of a disorder associated with elevated oxalateconcentrations. The effective amount can be determined by methods wellknown in the art and as described in subsequent sections of thisdescription.

The terms “treatment,” “therapeutic method,” and their cognates refer totreatment of an existing disorder and/or prophylactic/preventativemeasures. Those in need of treatment may include individuals alreadyhaving a particular medical disorder, as well as those at risk orhaving, or who may ultimately acquire the disorder. The need fortreatment is assessed, for example, by the presence of one or more riskfactors associated with the development of a disorder, the presence orprogression of a disorder, or likely receptiveness to treatment of asubject having the disorder. Treatment may include slowing or reversingthe progression of a disorder.

Oxalate Decarboxylase

As used herein, oxalate decarboxylase (OXDC) (EC 4.1.1.2) refers to anoxalate carboxy-lyase enzyme. Oxalate decarboxylases are a group ofenzymes known in the art to be capable of catalyzing the molecularoxygen (O₂) independent oxidation of oxalate to carbon dioxide andformate according to the following reaction:

HO₂C—CO₂H→1CO₂+HCOOH

Isoforms of oxalate decarboxylase, and glycoforms of those isoforms, areincluded within this definition. OXDC from plants, bacteria and fungiare encompassed by the term, including the true oxalate decarboxylasesfrom bacteria and fungi, such as Bacillus subtilis, Collybia velutipesor Flammulina velutipes, Aspergillus niger, Psoudomonas sp.,Synechocystis sp., Streptococcus nutans, Trametes hirsute, Scierotiniasclerotiorum, T. versicolor, Postia placenta, Myroth verrucaria,Agaricus bisporus, Methylobacterium extorquens, Pseudomonas oxalaticus,Ralstonia eutropha, Cupriavidus oxalaticus, Wautersia sp.,Oxalicibacterium flavum, Ammoniiphilus oxalaticus, Vibrio oxalaticus, A.oxalativorans, Variovorax paradoxus, Xanthobacter autotrophicus,Aspergillus sp., Penicillium sp., and Mucor species. Optionally, theOXDC will be additionally dependent on coenzyme A, such as OXDC fromorganisms in the intestinal tract. In certain circumstances, OXDC is asoluble or insoluble hexameric protein.

Oxalate decarboxylases used to prepare the crystals, and which are usedin methods described herein, may be isolated, for example, from anatural source, or may be derived from a natural source. As used herein,the term “derived from” means having an amino acid or nucleic acidsequence that naturally occurs in the source. For example, oxalatedecarboxylase derived from Bacillus subtilis will comprise a primarysequence of a Bacillus subtilis oxalate decarboxylase protein, or willbe encoded by a nucleic acid comprising a sequence found in Bacillussubtilis that encodes an oxalate decarboxylase or a degenerate thereof.A protein or nucleic acid derived from a source encompasses moleculesthat are isolated from the source, recombinantly produced, and/orchemically synthesized or modified. The crystals provided herein may beformed from polypeptides comprising amino acid sequences of OXDC or froma functional fragment of OXDC that retains oxalate degrading activity.Preferably, the OXDC retains at least one functional characteristic of anaturally occurring OXDC, e.g., the ability to catalyze degradation ofoxalate, the ability to multimerize, and/or manganese requirement.

Isolated Oxalate Decarboxylase

Oxalate decarboxylases have been previously isolated and are thusavailable from many sources, including Bacillus subtilis, Collybiavelutipes or Flammulina velutipes, Aspergillus niger, Pseudomonas sp.,Synechocystis sp., Streptococcus mutans, Trametes hirsute, Sclerotiniasclerotiorum, T. versicolor, Postia placenta, Myrothecium verrucaria,Agaricus bisporus, Methylobacterium oxtorqueris, Pseudomonas oxalaticus,Ralstonia eutropha, Cupriavidus oxalaticus, Wautersia sp.,Oxalicibacterium flavum, Ammoniiphilus oxalaticus, Vibrio oxalaticus, A.oxalativorans, Variovorax paradoxus, Xanthobacter autotrophicus,Aspergillus sp., Penicillium sp., and Mucor species. OXDC may also bepurchased from commercial purveyors, such as, e.g., Sigma. Methods toisolate OXDC from a natural source are previously described, forexample, in the following references: Tanner et al., J. Biol. Chem.47:43627-43634 (2001); Dashek and Micales, Methods in plant biochemistiyand molecular biology Boca Raton, Fla.: CRC Press 5:49-71 (1997); Magroet al., FEMS Microbiology Letters 49: 49-52 (1988); Anand et al.,Biochemistry 41:7659-7669 (2002); and Tanner and Bornemann, J.Bacteriol. 182: 5271-5273 (2000). These isolated oxalate decarboxylasesmay be used to form the crystals and methods described herein.

Recombinant Oxalate Decarboxylase

Alternatively, recombinant OXDCs may be used to form the crystals andmethods provided herein, In some instances, recombinant OXDCs encompassor are encoded by sequences from a naturally occurring OXDC sequence.Further, OXDCs comprising an amino acid sequence that is homologous orsubstantially identical to a naturally occurring sequence are hereindescribed. Also, OXDCs encoded by a nucleic acid that is homologous orsubstantially identical to a naturally occurring OXDC-encoding nucleicacid are provided and may be crystallized and/or administered asdescribed herein.

Polypeptides referred to herein as “recombinant” are polypeptides whichhave been produced by recombinant DNA methodology, including those thatare generated by procedures which rely upon a method of artificialrecombination, such as the polymerase chain reaction (PCR) and/orcloning into a vector using restriction enzymes.

“Recombinant” polypeptides also include polypeptides having alteredexpression, such as a naturally occurring polypeptide with recombinantlymodified expression in a cell, such as a host cell.

In one embodiment, OXDC is recombinantly produced from a nucleic acidthat is homologous to a Bacillus subtilis or Collybia veiutipes OXDCnucleic acid sequence, and sometimes it is modified, e.g., to increaseor optimize recombinant production in a heterologous host. An example ofsuch a modified sequence includes the nucleic acid sequence of the openreading frame of Collybia velutipes OXDC, for expression in Candidaboldinii. The OXDC sequence may be modified to reduce its GC content, tobe linked to a secretion signal sequence, e.g., an a Mating Factorsecretion signal sequence, and/or to be flanked by engineeredrestriction endonuclease cleavage sites. In another embodiment, OXDC isrecombinantly produced or from the unmodified Bacillus subtilis OXDCnucleic acid sequence which is available at GenBank Accession No:Z99120.The amino acid sequence encoded by this unmodified Bacillus subtilisOXDC nucleic acid sequence is provided as SEQ ID NO:1 as shown below.

  1 MKKQNDIPQPIRGDKGATVKIPRNIERDRQNPDMLVPPETDHGTVSNMK  50FSFSDTHNRLEKGGYAREVTVRELPISENLASVNMRLKPGAIRELHWHKE 100AEWAYMIYGSARVTIVDEKGRSFIDDVGEGDLWYFPSGLPHSIQALEEGA 150EFLLVFDDGSFSENSTFQLTDWLAHTPKEVlAANFGVTKEEISNLPGKEK 200YIFENQLPGSLKDDlVEGPNGEVPYPETYRLLEQEPIESEGGKVYIADST 250NFKVSKTIASALVTVEPGAMRELHWHPNTHEWQYYISGKARMTVFASDGH 300ARTFNYQAGDVGYVPFAMGHYVENIGDEPLVFLEIFKDDHYADVSLNQWL 350AMLPETFVQAHLDLGKDFTDVLSKEKHPVVKKKCSK 385

OXDC polypeptides useful for forming OXDC crystals may be expressed in ahost cell, such as a host cell comprising a nucleic acid construct thatincludes a coding sequence for an OXDC polypeptide or a functionalfragment thereof. A suitable host cell for expression of OXDC may beyeast, bacteria, fungus, insect, plant, or mammalian cell, for example,or transgenic plants, transgenic animals or a cell-free system. In someembodiments, a host cell is capable of glycosylating the OXDCpolypeptide if necessary, capable of disulfide linkages, capable ofsecreting the OXDC, andlor capable of supporting multimerization of OXDCpolypeptides. Preferred host cells include, but are not limited to E.coli (including E. coli Origami B and E. coli BL21), Pichia pastoris,Saccharomyces cerevisiae, Schizosaccharomyces pombe, Bacillus subtilis,Aspergillus, Sf9 cells, Chinese hamster ovary (CHO), 293 cells (humanembryonic kidney), and other human cells. Also transgenic plants,transgenic animals including pig, cow, goat, horse, chicken, and rabbitare suitable hosts for production of OXDC.

For recombinant production of OXDC, a host or host cell may comprise aconstruct in the form of a plasmid, vector, phagemid, or transcriptionor expression cassette that comprises at least one nucleic acid encodingan OXDC or a functional fragment thereof. A variety of constructs areavailable, including constructs which are maintained in single copy ormultiple copy, or which become integrated into the host cell chromosome.Many recombinant expression systems, components, and reagents forrecombinant expression are commercially available, for example fromInvitrogen Corporation (Carlsbad, Calif.); U.S. Biological (Swampscott,Mass.); BD Biosciences Pharrningen (San Diego, Calif.); Novagen(Madison, Wis.); Stratagene (La Jolla, Calif.); and Deutsche Sammlungvon Mikroorganismen and Zellkulturen GmbH (DSMZ), (Braunschweig,Germany).

Recombinant expression of OXDC is optionally controlled by aheterologous promoter, including a constitutive and/or induciblepromoter. Promoters such as, e.g., T7, the alcohol oxidase (AOX)promoter, the dihydroxy-acetone synthase (DAS) promoters, the Gal 1,10promoter, the phosphoglycerate kinase promoter, theglyceraldehyde-3-phosphate dehydrogenase promoter, alcohol dehydrogenasepromoter, copper metallothionein (CUP1) promoter, acid phosphatasepromoter, CMV and promoters polyhedrin are also appropriate. Theparticular promoter is selected based on the host or host cell. Inaddition, promoters that are inducible by methanol, copper sulfate,galactose, by low phosphate, by alcohol, e.g., ethanol, for example, mayalso be used and are well known in the art.

A nucleic acid that encodes OXDC may optionally comprise heterologoussequences. For example, a secretion sequence is included at theN-terminus of an OXDC polypeptide in some embodiments. Signal sequencessuch as those from a Mating Factor, BGL2, yeast acid phosphatase (PHO),xylanase, alpha amylase, from other yeast secreted proteins, andsecretion signal peptides derived from other species that are capable ofdirecting secretion from the host cell may be useful. Similarly otherheterologous sequences such as linkers (e.g., comprising a cleavage orrestriction endonuclease site) and one or more expression controlelements, an enhancer, a terminator, a leader sequence, and one or moretranslation signals are within the scope of this description. Thesesequences may optionally be included in a construct and/or linked to thenucleic acid that encodes OXDC. Unless otherwise specified, “linked”sequences can be directly or indirectly associated with one another.

Similarly, an epitope or affinity tag such as Histidine, HA(hemagglutinin peptide), maltose binding protein, AviTag®, FLAG, orglutathione-S-transferase may be optionally linked to the OXDCpolypeptide. A tag may be optionally cleavable from the OXDC after it isproduced or purified. A skilled artisan can readily select appropriateheterologous sequences, for example, match host cell, construct,promoter, and/or secretion signal sequence.

OXDC homologs or variants differ from an OXDC reference sequence by oneor more residues. Structurally similar amino acids can be substitutedfor some of the specified amino acids, for example. Structurally similaramino acids include: (I, L and V); (F and Y); (K and R); (Q and N); (Dand E); and (G and A). Deletion, addition, or substitution of aminoacids is also encompassed by the OXDC homologs described herein. Suchhomologs and variants include (i) polymorphic variants and natural orartificial mutants, (ii) modified polypeptides in which one or moreresidues is modified, and (iii) mutants comprising one or more modifiedresidues.

An OXDC polypeptide or nucleic acid is “homologous” (or is a “homolog”)if it is at least 40%,50%,60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99%, or 100% identical to a reference sequence. If the homolog is notidentical to the reference sequence, it is a “variant” A homolog is“substantially identical” to a reference OXDC sequence if the nucleotideor amino acid sequence of the homolog differs from the referencesequence (e.g., by truncation, deletion, substitution, or addition) byno more than 1, 2, 3, 4, 5, 8, 10, 20, or 50 residues, and retains (orencodes a polypeptide that retains) the ability to catalyze thedegradation of oxalate. Fragments of an oxalate decarboxylase may behomologs, including variants and/or substantially identical sequences.By way of example, homologs may be derived from various sources of OXDC,or they may be derived from or related to a reference sequence bytruncation, deletion, substitution, or addition mutation. Percentidentity between two nucleotide or amino acid sequences may bedetermined by standard alignment algorithms such as, for example, BasicLocal Alignment Tool (BLAST) described in Altschul et al., J Mol. Biol.,215:403 410 (1990), the algorithm of Needleman et al., J. Mol. Biol.,48:444 453 (1970), or the algorithm of Meyers et al., Comput. Appl.Biosci. 4:11-17 (1988). Such algorithms are incorporated into theBLASTN, BLASTP, and “BLAST 2 Sequences” programs (reviewed in McGinnisand Madden, Nucleic Acids Res. 32:W20-W25, (2004)). When utilizing suchprograms, the default parameters can be used. For example, fornucleotide equences the following settings can be used for “BLAST 2Sequences”: program BLASTN, reward for match 2, penalty for mismatch 2,open gap and extension gap penalties 5 and 2 respectively, gap x_dropoff50, expect 10, word size 11, filter ON, For amino acid sequences thefollowing settings can be used for “BLAST 2 Sequences”: program BLASTP,matrix BLOSUM62, open gap and extension gap penalties 11 and 1respectively, gap x_dropoff50, expect 10, word size 3, filter ON. Theamino acid and nucleic acid sequences for OXDCs that are appropriate toform the crystals described herein may include homologous, variant, orsubstantially identical sequences. In some embodiments, an OXDC homologretains at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99%, or 100% activity relative to a reference sequence.

Oxylate Decarboxylase Cysteine Modification

Without wishing to be bound by theory, thiol protection of C383 orelimination of the cysteine residue altogether, may enhance theformation of active oxalate decarboxylase hexamers, preventing oxidativedimerization among other oligomers. See, e.g., Tanner et al., J. Biol.Chem. 276(47):43627-34 (2001). Thiol protection or elimination of thecysteine residue of oxalate decarboxylase allows the protein to be morereadily processed into crystalline form for increased potency. To reduceproblems that may impact commercial scale production of oxalatedecarboxylase crystals, the C383 residue may be modified by substitutionof the amino acid as described in U.S. Pat. No. 8,431,122 or by deletionof C383. Alternatively, the thiol group of C383 of the oxalatedecarboxylase may be modified post-translationally with a thiolprotecting group to prevent it from reacting with other groups. Thiolprotecting groups are well-known to those skilled in the art and aredescribed, for example, in Greene and Wuts, Protecting Groups in OrganicSynthesis, Third Edition, Wiley, N.Y., (1999), and references citedtherein. For example, the thiol group of C383 may be protected byconverting it to a thioether, such as, e.g., an alkyl thioether, benzyland substituted benzyl thioether, triphenylmethyl thioether, or silylthioether; thioester; disulfide; thiocarbonate; or thiocarbamate.Alternatively, the thiol group of the C383 residue may be protected byadding a terminal cysteine, allowing the formation of an intramoleculardisulfide bridge to prevent the cysteine from reacting with othermolecules. In certain embodiments, the thiol group of C383 is protectedby cysteinylation. The invention provides crosslinked and/oruncrosslinked crystals of oxalate decarboxylase modified by, forexample, (1) elimination of C383, (2) addition of a C-terminal cysteine,or (3) reaction with a thiol protecting group by the invention(including cysteinylation) as well as compositions comprisingspray-dried OXDC crystals bearing one of these modifications.

Purification of Oxalate Decarboxylase

Oxalate decarboxylase proteins or polypeptides may be purified from thesource, such as a natural or recombinant source, prior tocrystallization. A polypeptide that is referred to herein as “isolated”is a polypeptide that is substantially free of its natural environment,such as proteins, lipids, and/or nucleic acids of their source of origin(e.g., cells, tissue (i.e., plant tissue), or fluid or medium (in thecase of a secreted polypeptide). Isolated polypeptides include thoseobtained by methods described herein or other suitable methods, andinclude polypeptides that are substantially pure or essentially pure,and polypeptides produced by chemical synthesis, by recombinantproduction, or by combinations of biological and chemical methods.Optionally, ani isolated protein has undergone further processing afterits production, such as by purification steps.

Purification may comprise buffer exchange and chromatographic steps,Optionally, a concentration step may be used, e.g., by dialysis,chromatofocusing chromatography, and/or associated with buffer exchange.In certain instances, cation or anion exchange chromatography is usedfor purification, including Q-sepharose, DEAF sepharose, DE52,sulfopropyl Sepharose chromatography or a CM52 or similar cationexchange column. Buffer exchange optionally precedes chromatographicseparation, and may be performed by tangential flow filtration such asdiafiltration. In certain preparations, OXDC is at least 70%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.7%, or 99.9% pure.

Purification in gram-scale runs is appropriate to prepare OXDC, andprocedures are optimized for efficient, inexpensive, manufacturing-scaleOXDC purification. For example, purification of at least 0.5, 1, 2, 5,10, 20, 50, 100, 500, or 1000 grams or more of OXDC in a purificationprocedure is provided. In one exemplary procedure, tangential flowfiltration of starting samples of at least 10 L, 50 L, 100 L, 500 L,1000 L or more is provided, allowing buffer exchange and precipitationof contaminant proteins. A single Q-sepharose column is optionally usedfor purification of OXDC.

Crystallization of Oxalate Decarboxylase

Oxalate decarboxylase crystals can be prepared using an OXDCpolypeptide, such as a hexamer, as described above. See Anand et al.,Biochemistry 41:7659-7669 (2002)). Vapor diffusion (such as, e.g.,hanging drop and sitting drop methods), and batch methods ofcrystallization, for example, can be used. Oxalate decarboxylasecrystals may be grown by controlled crystallization of the protein outof an aqueous solution or an aqueous solution that includes organicsolvents. Conditions to be controlled include the rate of evaporation ofsolvent, the presence of appropriate co-solutes and buffers, pH, andtemperature, for example.

For therapeutic administration, such as to treat a condition or disorderrelated to oxalate levels, a variety of OXDC crystal sizes areappropriate. In certain embodiments, crystals of less than about 500 μmaverage dimension are administered. Oxalate decarboxylase crystals withan average, maximal, or minimal dimension (for example) that is about0.01, 0.1, 1, 5, 10, 25, 50, 100, 200, 300, 400, 500, or 1000 μm inlength are also provided. Microcrystalline showers are also suitable.

Ranges are appropriate and would be apparent to the skilled artisan. Forexample, the protein crystals may have a longest dimension between about0.01 μm and about 500 μm, alternatively, between 0.1 μm and about 50 μm.In a particular embodiment, the longest dimension ranges from about 0.1μm to about 10 μm. Crystals may also have a shape chosen from spheres,needles, rods, plates, such as hexagons and squares, rhomboids, cubes,bipyramids and prisms. In illustrative embodiments, the crystals arecubes having a longest dimension of less than 5 μm.

In general, crystals are produced by combining the protein to becrystallized with an appropriate aqueous solvent or aqueous solventcontaining appropriate crystallization agents, such as salts or organicsolvents. The solvent is combined with the protein and optionallysubjected to agitation at a temperature determined experimentally to beappropriate for the induction of crystallization and acceptable for themaintenance of protein activity and stability. The solvent canoptionally include co-solutes, such as monovalent or divalent cations,co-factors or chaotropes, as well as buffer species to control pH. Theneed for co-solutes and their concentrations are determinedexperimentally to facilitate crystallization. In an industrial scaleprocess, the controlled precipitation leading to crystallization can becarried out by the combination of protein, precipitant, co-solutes and,optionally, buffers in a batch process, for example. Alternativelaboratory crystallization methods and conditions, such as dialysis orvapor diffusion, can be adopted (McPherson, et al., Methods Enzymol.114:112-20 (1985) and Gilliland, Crystal Growth 90:51-59 (1998)).Occasionally, incompatibility between the cross-linking agent and thecrystallization medium might require changing the buffers (solvent)prior to cross-linking.

As set forth in the Examples, oxalate decarboxylase crystallizes under anumber of conditions, including a wide pH range (e.g., pH 3.5 to 8.0). Aprecipitant such as a polyethylene glycol (such as, e.g., PEG 200, PEG400, PEG 600, PEG 1000, PEG 2000, PEG 3000, PEG 8000) or an organiccosolvent such as 2-methyl-2,4-pentanedial (MPD) is included in someembodiments as described. Common salts that may be used include sodiumchloride, potassium chloride, ammonia sulfate, zinc acetate, etc.

Oxalate decarboxylase may be at a concentration of, e.g., at least 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 mg/mI, ormore in a crystallization broth. The efficiency or yield of acrystallization reaction is at least 50%, 60%, 70%, 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97% 98%, 99%, or more. In one embodiment, crystalsof oxalate decarboxylase are grown or produced by a batch process bymixing a solution of oxalate decarboxylase with an appropriate buffer.

Crystallization from Cells or Cell Extract

Crystals may be prepared directly from cells or crude cell extracts. Inone embodiment, bacteria cells expressing oxalate decarboxylase areharvested. Cells are resuspended with or without DNase and homogenized.A salt solution is added to the cell lysis to reach a salt concentrationof about 0.3 M, 0.4 M, 0.5 M, 0.6 M, up to 2.5 M, 3.0 M, 3.5 M, 4.0 M,or more. The salt added can be a sodium salt, a potassium salt, acalcium salt, or other salts, Proteins may be optionally extracted fromthe cell mixture by removing cell debris. In one embodiment, homogenizedcell mixture is centrifuged, leaving proteins in the supernatantsolution. Crystals are generated by reducing salt concentration of thecell mixture or protein solution. In one embodiment, salt is removedthrough dialysis to maintain protein concentration. To increase crystalyield, protein solution may be concentrated before salt concentration ofthe solution is reduced. Crystals may be generated at a solution with apH of about 6, 7, 8 or 9.

Crystals may be prepared from a protein precipitate or pellet. In oneembodiment, cells expressing desired proteins are harvested and oxalatedecarboxylase protein is collected in a precipitate or pellet. Pellet orprecipitate containing oxalate decarboxylase protein is solubilized in asalt solution. Crystals are formed by reducing salt concentration in theprotein solution. For increased crystal yields, the salt concentrationin the solubilized protein solution is at least about 0.3 M, 0.4 M, 0.5M or more before it is reduced to produce crystals.

Crystals may also be prepared from a protein solution. In oneembodiment, an oxalate decarboxylase protein solution is concentrated ina salt solution, and crystals are formed when the salt concentration inthe solution is reduced. For increased crystal yields, the saltconcentration is at least about 0.3 M, 0.4 M, 0.5 M or more before it isreduced to produce crystals.

Drying of Crystals of Oxalate Decarboxylase

Pharmaceutical proteins, including protein crystals may be dried in manyways, e.g., by removal of water, organic solvent or liquid polymer bymeans including drying with N₂, air or inert gases, vacuum oven drying,lyophilization, washing with a volatile organic solvent followed byevaporation of the solvent, evaporation in a fume hood, tray drying,fluid bed drying, spray drying, vacuum drying, or roller drying.Typically, tray drying, i.e. drying carried out by passing a stream ofgas over wet crystals is used to pharmaceutical proteins. The gas may beselected from the group consisting of: nitrogen, argon, helium, carbondioxide, air or combinations thereof. Drying is achieved when thecrystals become a free flowing powder.

Spray drying is rarely used to prepare pharmaceutical grade proteins dueto concerns related to sterility. No reports of spray-dried crystalshave been found in the literature. Prior to this invention, it waswidely believed that protein crystals could not be spray-dried becausethe crystals would clog the spray-dryer during the manufacturing processand that even if the spray-dryer could be adapted to process proteincrystals, the crystals themselves would not survive the process. It wasexpected that the crystals would be fractured and broken down to a dustduring processing, essentially elirninating any advantages associatedwith the crystalline form. Thus, it is surprising and unexpected thatpharmaceutical compositions of the invention comprise spray-driedoxalate decarboxylase crystals that retain their crystalline integrity,stability, and enzymatic activity, as well as providing advantagesassociated with spray-dried formulations.

Spray drying oxalate decarboxylase crystals allows water to be separatedfrom the crystal preparation, allowing for continuous production of drysolids in powder, granulate, or agglomerate form from liquid feedstockssuch as emulsions and pumpable suspensions. Solutions of crystals cannotbe used because once the crystals are dissolved, the crystals do notreform upon drying.

Spray drying involves the atomization of a liquid feedstock comprisingOXDC crystals into a spray of droplets and contacting the droplets withhot air or gas in a drying chamber. The atomization process is bestconducted using a two-fluid atomizer that mixes the liquid feedstockwith a drying gas such as compressed air or nitrogen. One-fluid androtary atomizers develop high-shear which could easily break thecrystals. Evaporation of moisture from the droplets and formation of dryparticles proceed under controlled temperature and airflow conditions.

Because of an evaporative cooling effect during the critical dryingperiod and because the time of exposure to high temperatures of the drymaterial is generally short, the process results in little damage to thecrystalline protein. Dried crystalline protein powder is dischargedcontinuously from the drying chamber. Operating conditions and dryerdesign are selected according to the drying characteristics of the OXDCcrystals and the desired powder qualities. The dried OXDC crystals arethen tested for compliance with quality standards regarding particlesize distribution, residual moisture content, bulk density and particleshape. In some embodiments excipients or ingredients selected fromsugars, sugar alcohols, viscosity increasing agents, wetting orsolubilizing agents, buffer salts, emulsifying agents, antimicrobialagents, antioxidants, water soluble polymers, amino acids and coatingagents are added directly to the liquid feedstocks prior to spraydrying. The excipient concentration is typically between about 10% andabout 50% (w/w). The crystal concentration is typically between about 5%and about 25% (w/w). Crystal suspensions with and without excipients canbe frozen at about −10° C. to about −70° C. for up to one year without adetrimental impact on the crystal structure or activity.

Compositions Comprising Spray-Dried OXDC Crystals

Spray-dried OXDC crystals are provided as a composition, such as apharmaceutical composition (see, e.g., U.S. Pat. No. 6,541,606,describing formulations and compositions of protein crystals).Pharmaceutical compositions comprising spray-dried OXDC crystals mayinclude one or more ingredients or excipients, including, but notlimited to sugars and biocompatible polymers. Examples of excipients aredescribed in Handbook of Pharmaceutical Excipients, published jointly bythe American Pharmaceutical Association and the Pharmaceutical Societyof Great Britain, and further examples are set forth below.

The OXDC enzyme may be administered as a spray-dried crystal in acomposition as any of a variety of physiologically acceptable saltforms, and/or with an acceptable pharmaceutical carrier and/or additiveas part of a pharmaceutical composition.

Physiologically acceptable salt forms and standard pharmaceuticalformulation techniques and excipients are well known to persons skilledin the art (see, e.g., Physician's Desk Reference (PDR) 2003, 57th ed.,Medical Economics Company, 2002; and Remington: The Science and Practiceof Pharmacy, eds. Gennado et al., 20th ed, Lippincott, Williams &Wilkins, 2000). For the purposes of this application, “formulations”include “crystal formulations.”

Oxalate decarboxylase useful in the methods of the present disclosuremay be combined with an excipient. According to the present disclosure,an “excipient” acts as a filler or a combination of fillers used inpharmaceutical compositions. Exemplary ingredients and excipients foruse in the compositions are set forth as follows.

Sugars

The sugar used as an excipient may be a monosaccharide, disaccharide,oligosaccharide, or polysaccharide. Exemplary monosaccharides includebut are not limited to ribose, arabinose, xylose, lyxose, allose,altrose, glucose, mannose, gulose, fructose, iodose, galactose, xylitol,sucralose and talose. Exemplary disaccharides include but are notlimited to sucrose, lactose, maltose, lactulose, trehalose, andcellobiose.

Biocompatible Polymers

Biocompatible polymers are polymers that are non-antigenic,non-carcinogenic, non-toxic and which are not otherwise inherentlyincompatible with living organisms may be used in the OXDC crystalcompositions described herein. Examples include: poly (acrylic acid),poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly(depsipeptide), poly (esters) such as poly (lactic acid) or PLA, poly(lactic-co-glycolic acid) or PLGA, poly (β-hydroxybutryate), poly(caprolactone) and poly (dioxanol); poly (ethylene glycol), poly((hydroxypropyl)methacrylamide, poly [(organo)phosphazene], poly (orthoesters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleicanhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin,alginate, cellulose and cellulose derivatives, collagen, fibrin,gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfatedpolysaccharides, blends and copolymers thereof.

Biodegradable Polymers

Biodegradable polymers degrade by hydrolysis or solubilization may beincluded in OXDC crystal compositions. Degradation can be heterogenous(occurring primarily at the particle surface), or homogenous (degradingevenly throughout the polymer matrix). Ingredients such as one or moreexcipients or pharmaceutical ingredients or excipients may be includedin OXDC crystal compositions. An ingredient may be an inert or activeingredient.

In some embodiments, the pharmaceutical composition comprisescrystallized oxalate decarboxylase which is spray-dried. In otherembodiments, an excipient is added to the crystallized oxalatedecarboxylase, and then the mixture is spray-dried to form thepharmaceutical composition. In certain ernboduments, the excipient is asugar. In certain embodiments, the sugar is a monosaccharide or adisaccharide. In some embodiments the excipient is trehalose, sucrose,or glucose. In certain embodiments the excipient is trehalose.

In an exemplary embodiment, trehalose is added to oxalate decarboxylasecrystals and the mixture is spray-dried to form a pharmaceuticalcomposition.

Flow Characteristics of Spray-Dried OXDC Crystals

According to the USP, the generally accepted scale of flowability isgiven in Table 1:

TABLE 1 Scale of Flowability Flow Character Hausner Ratio Excellent1.00-1.11 Good 1.12-1.18 Fair 1.19-1.25 Passable 1.26-1.34 Poor1.35-1.45 Very Poor 1.46-1.59 Very, very poor >1.60

In some embodiments, the flowability of the pharmaceutical compositionis “passable” or better per USP, i.e., the Hausner ratio is 1.34 orless. In some embodiments, the flowability of the pharmaceuticalcomposition is “fair” or better per USP, i.e., the Hausner ratio is 1.25or less. In further emodiments, the flowability of the pharmaceuticalcomposition is “good” or better per USP, i.e., the Hausner ratio is 1.18or less. In some embodiments the Hausner ratio of the pharmaceuticalcomposition is from about 1.12 to about 1.18.

In some embodiments the pharmaceutical composition exhibits betterstability than non-crystalline oxalate decarboxylase at a pH from about3.5 to about 8 or 9. In some embodiments the pharmaceutical compositionexhibits better stability than non-crystalline oxalate decarboxylase ata pH from about 3.5 to about 7. In some embodiments the pharmaceuticalcomposition exhibits better stability than non-crystalline oxalatedecarboxylase at a pH from about 3.5 to about 6. In some embodiments thepharmaceutical composition exhibits better stability thannon-crystalline oxalate decarboxylase at a pH from about 3.5 to about 5.In some embodiments, the pharmaceutical composition exhibits betterstability than soluble oxalate decarboxylase at a pH of about 3.7. Insome embodiments the pharmaceutical composition exhibits betterstability than non-crystalline oxalate decarboxylase in the presence ofpepsin, trypsin or chymotrypsin.

In some embodiments the pharmaceutical composition is filled intocapsules. In some embodiments the gelatin capsules are size 000, 00, 0,1 or 2 capsules. In some embodiments the pharmaceutical composition ispressed into tablets. In some embodiments the pharmaceutical compositionis blended to form a suspension. In some embodiments the capsules,tablets, or suspension comprising the pharmaceutical composition aresuitable for oral administration.

Methods of Treating Oxalate-Associated Disorders with Spray-Dried OXDCCrystals

The methods of the present disclosure comprise administering an oxalatedecarboxylase, e.g., spray-dried crystals of OXDC, or cross-linked formsthereof, to a mammalian subject to treat, prevent, or reduce the risk ofoccurrence of a condition associated with elevated levels of oxalate.The elevated levels of oxalate may be detected, e.g., in a biologicalsample from the subject, such as a body fluid, including urine, blood,serum, or plasma In certain embodiments, urinary oxalate levels aredetected. The crystals and/or the compositions comprising crystals maybe administered in the methods described herein.

In some embodiments, methods are provided for treating hyperoxaluria inindividuals with primary hyperoxaluria, enteric hyperoxaluria,hyperoxaluria caused by surgical intervention, idiopathic hyperoxaluria,oxalosis are provided. In other instances, elevated oxalate-relateddisorders of the kidneys, bone, liver gastrointestinal tract andpancreas are amenable to treatment with the methods disclosed herein.Further disorders or diseases treated by the methods provided hereininclude, but are not limited to ethylene glycol (oxalate) poisoning,idiopathic urinary stone disease, renal failure (including progressive,chronic, or end-stage renal failure), steatorrhoea, malabsorption, ilealdisease, vulvodynia, cardiac conductance disorders, inflammatory boweldisease, cystic fibrosis, exocrine pancreatic insufficiency, Crohn'sdisease, ulcerative colitis, nephrocalcinosis, osteoporosis,urolithiasis, and nephrolithiasis. Such conditions and disorders mayoptionally be acute or chronic.

The methods of the present disclosure may reduce oxalate levels in asubject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, ormore as compared to levels in an untreated or control subject. In someembodiments, reduction is measured by comparing the oxalate level in asubject before and after administration of OXDC. In some embodiments,the present disclosure provides a method of treating or ameliorating anoxalate-associated condition or disorder, to allow one or more symptomsof the condition or disorder to improve by at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95% or more. In certain embodiments the methodsreduce levels of endogenous oxalate and/or adsorption of dietaryoxalate.

In some embodiments, methods for treating individuals having a genotypeassociated with high oxalate levels are provided, such as individualshomozygous or heterozygous for a mutation that reduces activity of,e.g., alanine:glyoxalate aminotransferase, glyoxylatereductase/hydroxypyruvate reductase, hepatic glycolate oxidase, oranother enzyme involved in oxalate metabolism or associated withhyperoxaluria. In other embodiments, methods for treating individualshaving reduced or lacking Oxalobacter formigenes enteric colonizationare provided.

The disclosed methods include administering therapeutically effectiveamounts of oxalate decarboxylase to a mammalian subject at risk for,susceptible to, or afflicted with a condition associated with elevatedlevels of oxalate. The populations treated by the methods of the presentdisclosure include, but are not limited to, subjects suffering from, orat risk for developing an oxalate-associated disorder such as, e.g.,primary hyperoxaluria or enteric hyperoxaluria.

Subjects treated according to the methods of the present disclosureinclude but are not limited to mammals, including humans, non humanprimates, primates, baboons, chimpanzees, monkeys, rodents (e.g., mice,rats), rabbits, cats, dogs, horses, cows, sheep, goats, pigs, etc.

Indication, Symptoms, and Disease Indicators

Many methods are available to assess development or progression of anoxalate-associated disorder or a condition associated with elevatedoxalate levels. Such disorders include, but are not limited to, anycondition, disease, or disorder as defined above. Development orprogression of an oxalate-associated disorder may be assessed bymeasurement of urinary oxalate, plasma oxalate, measurement of kidney orliver function, or detection of calcium oxalate deposits, for example.

A condition, disease, or disorder may be identified by detecting ormeasuring oxalate concentrations, for example, in a urine sample orother biological sample or fluid. An early symptom of hyperoxaluria istypically kidney stones, which may be associated with severe or suddenabdominal or flank pain, blood in the urine, frequent urges to urinate,pain when urinating, or fever and chills. Kidney stones may besymptomatic or asymptomatic, and may be visualized, for example byimaging the abdomen by x-ray, ultrasound, or computerized tomography(CT) scan. if hyperoxaluria is not controlled, the kidneys are damagedand kidney function is impaired. Kidneys may even fail.

Kidney failure (and poor kidney function) may be identified by adecrease in, or lacking urine output (glomerular filtration rate),general ill feeling, tiredness, and marked fatigue, nausea, vor itirig,anemia, and/or failure to develop and grow normally in young children.

Calcium oxalate deposits in other tissues and organs may also bedetected by methods including direct visualization (e.g. in the eyes),x-ray, ultrasound, CT, echocardiogram, or biopsy (e.g., bone, liver, orkidney). Kidney and liver function, as well as oxalate concentrations,may also be assessed using art-recognized direct and indirect assays.The chemical content of urine, blood or other biological sample may alsobe tested by well known techniques. For example, oxalate, glycolate, andglycerate levels may be measured. Assays for liver and kidney functionare well known, such as, for example, the analysis of liver tissue forenzyme deficiencies and the analysis of kidney tissue for oxalatedeposits. Samples may also be tested for DNA changes known to causeprimary hyperoxaluria.

Other indications for treatment include, but are not limited to, thepresence of one or more risk factors, including those discussedpreviously and in the following sections. A subject at risk fordeveloping or susceptible to a condition, disease, or disorder or asubject who may be particularly receptive to treatment with oxalatedecarboxylase may be identified by ascertaining the presence or absenceof one or more such risk factors, diagnostic, or prognostic indicators.Similarly, an individual at risk for developing an oxalate-relateddisorder may be identified by analysis of one or more genetic orphenotypic markers.

The methods disclosed are useful in subjects with urinary oxalate levelsof at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 mg of oxalateper 24 hour period, or more. In certain embodiments, the oxalate levelis associated with one or more symptoms or pathologies. Oxalate levelsmay be measured in a biological sample, such as a body fluid includingblood, serum, plasma, or urine. Optionally, oxalate is normalized to astandard protein or substance, such as creatinine in urine. In someembodiments, the claimed methods include administration of oxalatedecarboxylase to reduce circulating oxalate levels in a subject toundetectable levels, or to less than 1%, 2%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, or 80% of the subject's oxalate levels prior totreatment, within 1, 3, 5, 7, 9, 12, or 15 days.

Hyperoxaluria in humans can be characterized by urinary oxalateexcretion of greater than 40 mg (approximately 440 μmol) or 30 mg perday. Exemplary clinical cutoff levels are 43 mg/day (approximately 475μmol) for men and 32 mg/day (approximately 350 μmol) for women, forexample. Hyperoxaluria can also be defined as urinary oxalate excretiongreater than 30 mg per day per gram of urinary creatinine. Persons withmild hyperoxaluria may excrete at least 30-60 (342-684 pmol) or 40-60(456-684 μmol) mg of oxalate per day. Persons with enteric hyperoxaluriamay excrete at least 80 mg of urinary oxalate per day (912 μmol), andpersons with primary hyperoxaluria may excrete at least 200 mg per day(2280 μmol).

Administration of Spray-Dried OXDC Crystals and Compositions

Administration of oxalate decarboxylase in accordance with the methodsof the present disclosure is not limited to any particular deliverysystem and includes administration via the upper gastointestinal tract,e.g., the mouth (for example in capsules, liquid suspension, tablets, orwith food), or the stomach, or upper intestine (for example by tube ornjection) to reduce oxalate levels in an individual. In certain cases,the OXDC is administered to reduce endogenous oxalate levels and/orconcentrations, OXDC may also be provided by an extracorporeal device,such as a dialysis apparatus, a catheter, or a structure or device thatcontacts a biological sample from an individual.

Administration to an individual may occur in a single dose or in repeatadministrations, and in any of a variety of physiologically acceptableforms, and/or with an acceptable pharmaceutical carrier and/or additiveas part of a pharmaceutical composition (described earlier). In thedisclosed methods, oxalate decarboxylase may be administered alone,concurrently or consecutively over overlapping or nonoverlappingintervals with one or more additional biologically active agents, suchas, e.g., pyridoxine (vitamin B-6), orthophosphate, magnesium,glycosaminoglycans, calcium, iron, aluminum, magnesium, potassiumcitrate, cholestyramine, organic marine hydrocolloid, plant juice, suchas, e.g., banana stem juice or beet juice, or L-cysteine. Biologicallyactive agents that reduce oxalate levels or that increase the activityor availability of OXDC are provided. In sequential administration, theoxalate decarboxylase and the additional agent or agents may beadministered in any order. In some embodiments, the length of anoverlapping interval may be more than 2 4. 6, 12, 24, or 48 weeks ormore.

The oxalate decarboxylase may be administered as the sole activecompound or in combination with another active compound or composition.Unless otherwise indicated, the oxalate decarboxylase is administered asa dose of approximately from 10 μg/kg to 25 mg/kg or 100 mg/kg,depending on the severity of the symptoms and the progression of thedisease. The appropriate therapeutically effective dose of OXDC isselected by a treating clinician and would range approximately from 10μg/kg to 20 mg/kg, from 10 μg/kg to 10 mg/kg, fro 10 μg/kg to 1 mg/kg,from 10 pg/kg to 100 μg/kg , from 100 μg/kg to 1 mg/kg, from 100 μg/kgto 10 mg/kg, from 500 μg/kg to 5 mg/kg, from 500 μg/kg to 20 mg/kg, from1 mg/kg to 5 mg/kg, from 1 mg/kg to 25 mg/kg, from 5 mg/kg to 100 mg/kg,from mg/kg to 50 mg/kg, from 5 mg/kg to 25 mg/kg, and from 10 mg/kg to25 mg/kg. Additionally, specific dosages indicated in the Examples or inthe Physician's Desk Reference (PDR) 2003, 57th ed., Medical EconomicsCompany, 2002, may be used.

In some embodiments the dosage of oxalate decarboxylase is about 900,about 3600, about 10800, or about 18000 units oxalate decarboxylase. Insome embodiments the dosage is about 1500 units oxalate decarboxylaseper capsule. In some embodiments the dosage of oxalate decarboxylase isabout 112, about 450, about 1350, or about 2250 mg oxalatedecarboxylase. In some embodiments the dosage is about 190 mg oxalatedecarboxylase per capsule. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, or 15 capsules are administered to a mammal, suchas a human, per day. In some embodiments the capsules are administeredwith food, such as a snack or meal.

The oxalate decarboxylase crystal of the present disclosure may beadministered through an extracorporeal device or catheter, such as fordelivery of oxalate decarboxylase to a patient. Catheters, for example,urinary catheters, may be coated with compositions containing oxalatedecarboxylase crystals.

In some embodiments, the oxalate decarboxylase crystals retain at least50%, 60%, 70%, 80%, 90%, 95%, or 99% of their activity after 120 minutesat pH 9. In some embodiments, the oxalate decarboxylase crystals retainat least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of their activity after120 minutes at pH 8. In some embodiments, the oxalate decarboxylasecrystals retain at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of theiractivity after 120 minutes at pH 7. In some embodiments, the oxalatedecarboxylase crystals retain at least 50%, 60%, 70%, 80%, 90%, 95%, or99% of their activity after 120 minutes at pH 6. In some embodiments,the oxalate decarboxylase crystals retain at least 50%, 60%, 70%, 80%,90%, 95%, or 99% of their activity after 120 minutes at pH 5. In someembodiments, the oxalate decarboxylase crystals retain at least 50%,60%, 70%, 80%, 90%, 95%, or 99% of their activity after 120 minutes atpH 4. In some embodiments, the oxalate decarboxylase crystals retain atleast 50%, 60%, 70%, 80%, 90%, 95%, or 99% of their activity after 60mnutes at pH 7. In some embodiments, the oxalate decarboxylase crystalsretain at least 50%, 60%, 70%, 80%. 90%, 95%, or 99% of their activityafter 60 minutes at pH 6. In some embodiments, the oxalate decarboxylasecrystals retain at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of theiractivity after 60 minutes at pH 5. In some embodiments, the oxalatedecarboxylase crystals retain at least 50%, 60%, 70%, 80%, 90%, 95%, or99% of their activity after 60 minutes at pH 4. In some embodiments, theoxalate decarboxylase crystals retain at least 50%, 60%, 70%, 80%, 90%,95%, or 99% of their activity after 30 minutes at pH 7. In someembodiments, the oxalate decarboxylase crystals retain at least 50%,60%, 70%, 80%, 90%, 95%, or 99% of their activity after 30 minutes at pH6. In some embodiments, the oxalate decarboxylase crystals retain atleast 50%, 60%, 70%, 80%, 90%, 95%, or 99% of their activity after 30minutes at pH 5. In some embodiments, the oxalate decarboxylase crystalsretain at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of their activityafter 30 minutes at pH 4.

In some embodiments, the oxalate decarboxylase crystals retain at least50%, 60%, 70%, 80%, 90%, 95%, or 99% of their activity after 120 minutesin simulated gastric fluid (SGF; pH 3.7) with pepsin. In someembodiments, the oxalate decarboxylase crystals retain at least 50%,60%, 70%, 80%, 90%, 95%, or 99% of their activity after 60 minutes insimulated gastric fluid (SGF; pH 3.7) with pepsin. In some embodiments,the oxalate decarboxylase crystals retain at least 50%, 60%, 70%, 80%,90%, 95%, or 99% of their activity after 30 minutes in simulated gastricfluid (SGF; pH 3.7) with pepsin.

In some embodiments the spray-dried crystals retain at least 50%, 60%,70%, or 80%, of their activity after 3 weeks at 40° C. and 75% RH. Insome embodiments the spray-dried crystals retain at least 50%, 60%, 70%,80%, or 85% of their activity after 2 weeks at 40° C. and 75% RH. insome embodiments the spray-dried crystals retain at least 50%, 60%. 70%.80%, or 90% of their activity after 1 week at 40° C. and 75% RH.

In some embodiments the spray-dried crystals with trehalose retain atleast 50%, 60%, 70%, 80%, 90%, or 100% of their activity after 3 weeks.In some embodiments the spray-dried crystals with trehalose retain atleast 50%, 60%, 70%, 80%, 90%, or 100% of their activity after 2 weeks.In some embodiments the spray-dried crystals with trehalose retain atleast 50%, 60%, 70%, 80%, 90%, or 100% of their activity after 1 week.In some embodiments the spray-dried crystals with sucrose retain atleast 50%, 60%, 70%, 80%, 90%, or 100% of their activity after 3 weeks.In some embodiments the spray-dried crystals with sucrose retain atleast 50%, 60%, 70%, 80%, 90%, or 100% of their activity after 2 weeks.In some embodiments the spray-dried crystals with sucrose retain atleast 50%, 60%, 70%, 80%, 90%, or 100% of their activity after 1 week.In some embodiments the spray-dried crystals with Kolloidon VA64 retainat least 50%, 60%, 70%, 80%, 90%, or 100% of their activity after 3weeks. In some embodiments the spray-dried crystals with Kolloidon VA64retain at least 50%, 60%, 70%, 80%, 90%, or 100% of their activity after2 weeks. In some embodiments the spray-dried crystals with KolloidonVA64 retain at least 50%, 60%, 70%, 80%, 90%, or 100% of their activityafter 1 week. In some embodiments the spray-dried crystals withKolloidon 12PF retain at least 50%, 60%, 70%, 80%, 90%, or 100% of theiractivity after 3 weeks. In some embodiments the spray-dried crystalswith Kolloidon 12PF retain at least 50%, 60%, 70%, 80%, 90%, or 100% oftheir activity after 2 weeks, In some embodiments the spray-driedcrystals with Kolloidon 12PF retain at least 50%, 60%, 70%, 80%, 90%, or100% of their activity after 1 week.

The following examples provide illustrative embodiments of the presentdisclosure. One of ordinary skill in the art will recognize the numerousmodifications and variations that may be performed without altering thespirit or scope of the present disclosure. Such modifications andvariations are encompassed within the scope of the present disclosure.The Examples do not in any way limit the present disclosure.

EXAMPLES

Oxalate decarboxylase crystals are designed to be active in the stomachand will be administered in the fed-state at a stomach pH>4. Thefollowing in vitro studies demonstrated that the cross-linked andnon-cross-linked OXDC were comparable relative to activity andstability.

Example 1

OXDC specific activity at 37° C. as a function of pH is shown in FIG. 1.These results demonstrate that non-cross-linked OXDC (crystals) has thesame or higher specific activity than the crosslinked material (OXDCCLEC) over the pH ranges evaluated,

Example 2

FIG. 2 shows the percent of retained activity over time for the crystalOXDC. The materials were incubated at the indicated pH for up to twohours followed by pH adjustment to pH 4.0 for the activity measurement.

Example 3

FIG. 3 shows the retained enzymatic activity over time of OXDC crystalsor CLEC in simulated gastric fluid (SGF: pH 3.7) with pepsin. Thestability of OXDC crystals in SGF at pH 3.7 with added pepsin is equalto or better than the CLEC form.

Example 4

For spray-drying development, the OXDC crystals were tested in a 40°C./75% RH accelerated development stability study to promote fasterdegradation and allow rapid screening of multiple formulations andtechnologies. A comparison of the 40° C. stability for representativelyophilized and spray-dried OXDC crystals is shown in FIG. 4. These datashow that the spray drying process selected for the OXDC crystals has asuperior accelerated stability profile relative to the lyophilizationprocess.

The spray drying process also results in crystals encased in amorphousspheres of trehalose excipient that prevent the crystals from physicallyaggregating This yields crystals with improved power flowcharacteristics over the lyophilized crystals. Representative data forspray-dried lots are presented in Table 2 and demonstrate “good”flowability per USP while the data for lyophilized crystals demonstrates“very, very poor” flowability per USP.

TABLE 2 Bulk and Tap Densities, and Hausner Ratio (Flowability Index)for Spray Dried Crystals and Lyophilized Crystals Bulk Density TappedDensity Hausner Entry (g/mL) (g/mL) Ratio^(‡) Spray-dried Crystals 10.36 0.42 1.17 2 0.37 0.43 1.16 3 0.43 0.49 1.14 Lyophilized Crystals 40.35 0.69 1.97 ^(‡)The Hausner ratio range for “good” flowability is1.12 to 1.18; the Hausner ratio for “very, very poor” flowability arevalues greater than 1.6.

Because the spray-dried crystals are much less compressible than thelyophilized crystals, the spray-dried crystals have improvedpowder-handling properties and may be used to produce capsules, tablets,sachets or suspensions without the need for compounding and blendingoperations. This allows a larger number of units to be loaded intocapsules, tablets, sachets or suspensions relative to lyophilizedcrystals and allows less wastage of material when filling capsules,tablets, sachets or suspensions, as well as reduced cost, improvedstability and for the delivery of therapeutic doses without a largenumber of capsules to be used.

Example 5

Oxalate decarboxylase spray-dried with each of sucrose, trehalose,copovidone (Kollidon VA64), or providone (Kolloidon 12PF) in separateformulations was tested in a 40° C./75% RH accelerated developmentstability study for its relative activity to t=0 for three weeks. Theresults are shown in FIG. 5. Sugar-based excipient exhibited superiorstability relative to polymer-based excipients. Spray-dried OXDC withsugar-based excipients also exhibited superior stability relative tospray-dried OXDC with no excipients (data not shown).

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made without departing fromthe spirit and scope of the present disclosure. Accordingly, otherembodiments are within the scope of the following claims.

1. A pharmaceutical composition comprising a spray-dried oxalatedecarboxylase crystal. 2.-133. (canceled)